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Transition Metal Ion (Mn2+, Fe2+, Co2+ and Ni2+)-Doped Carbondots Synthesized via Microwave-Assisted Pyrolysis: A Potential Nanoprobe for Magneto-fluorescent Dual-Modality Bioimaging Sajid Abdul Rub Pakkath, Shashank Shankar Chetty, Selvarasu Praneetha, Arumugam Vadivel Murugan, Yogesh Kumar, Latha Periyasamy, Muthukamalam Santhakumar, Sudha Rani Sadras, and Kirankumar Santhakumar ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00943 • Publication Date (Web): 22 May 2018 Downloaded from http://pubs.acs.org on May 23, 2018

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ACS Biomaterials Science & Engineering

Transition Metal Ion (Mn2+, Fe2+, Co2+ and Ni2+)Doped Carbon-dots Synthesized via MicrowaveAssisted Pyrolysis: A Potential Nanoprobe for Magneto-fluorescent Dual-Modality Bioimaging Sajid Abdul Rub Pakkath, ‡† Shashank Shankar Chetty, ‡† Praneetha Selvarasu, † Arumugam Vadivel Murugan, *† Yogesh Kumar,§ Latha Periyasamy,§ Muthukamalam Santhakumar,# Sudha Rani Sadras# and Kirankumar Santhakumar₸ *Corresponding author. Email: [email protected], [email protected] †

Advanced Functional Nanostructured Materials Research Laboratory, Centre for Nanoscience

and Technology, Madanjeet School of Green Energy Technologies, Pondicherry University (A Central University), Dr. R. Venkataraman Nagar, Kalapet, Puducherry 605014, India. §Laboratory of Phytomedicine and Toxicology, Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University (A Central University), Dr. R. Venkataraman Nagar, Kalapet, Puducherry 605014, India. #Laboratory of Phytotherapy and Degenerative Diseases, Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University (A Central University), Dr. R. Venkataraman Nagar, Kalapet, Puducherry 605014, India.

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ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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₸ Zebrafish Genetics Laboratory, Department of Genetic Engineering, Sree Ramaswamy Memorial (SRM) Institute of Science and Technology, SRM Nagar, Kattankulathur 603203, Tamil Nadu, India. KEYWORDS: Transition metal ion-doped carbon dot (TMCD), microwave-assisted pyrolysis, natural extract, Fluorescence bioimaging, magnetic resonance imaging, T1-contrast nanoprobe. ABSTRACT: Heteroatom-doped carbon dots (C-dots) have captured widespread research interest owing to high fluorescence and biocompatibility for multimodal bioimaging applications. Here we exemplify a rapid, facile synthesis of ethylenediamine (EDA)functionalized transition metal ion (Mn2+, Fe2+, Co2+ and Ni2+)-doped C-dots via one-pot microwave (MW)-assisted pyrolysis at 800 W within 6 mins using Citrus limon (lemon) extract as carbon source. During MW-pyrolysis, the precursor extract undergoes simultaneous carbonization and doping of metal ions onto C-dots surface in the presence of EDA. The EDAfunctionalized transition metal ion-doped C-dots (i.e. Mn/C, Fe/C, Co/C and Ni/C-dots) are collectively termed as TMCDs. The water-soluble TMCDs exhibited a size of 3.2 ± 0.485 nm and were enriched with amino, oxo-functionalities and corresponding metal oxides traces on the surfaces as revealed from FTIR and XPS analyses. Interestingly, TMCDs demonstrated excitation wavelength-dependent emission with brighter photoluminescence (PL) at 460 nm. Compared to pristine C-dots with PL quantum yield (QY) of 48.31 % and fluorescence lifetime of 3.6 ns, the synthesized Mn/C, Fe/C, Co/C and Ni/C-dots exhibited PL QY of 35.71, 41.72, 75.07 and 50.84 % as well as enhanced fluorescence lifetime (τav) of 9.4, 8.6, 9.2 and 8.9 ns respectively. The TMCDs significantly exhibited enhanced biocompatibility in human colon cancer cells (SW480) for fluorescence bioimaging and showed ferromagnetic/superparamagnetic behaviour with vibrant T1-contrast ability. Interestingly, the maximum longitudinal (r1) relaxivity


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of 0.341 mM-1s-1 was observed for Mn/C-dots in comparison to that of 3.1-3.5 mM-1s-1 of clinically used Gd-DTPA magnetic resonance (MR) contrast agent in vitro (1.5 T). Similarly, maximum longitudinal relaxivity (r1) of 0.356 mM-1s-1 was observed for Ni/C-dots (1.5 T) with respect to 4.16 ± 0.02 mM-1s-1 attained for Gd-DTPA in vivo (8.45 T). Thus the rapid, energyefficient MW-assisted pyrolysis presents lemon extract-derived, EDA-functionalized TMCDs with enhanced PL and efficient T1-contrast as potential magneto-fluorescent nanoprobes for dual-modality bioimaging applications. INTRODUCTION Carbon dots (C-dots) are regarded as an exquisite class of quasi-spherical, zero-dimensional fullerene-based carbonaceous photonic nanomaterials with size < 10 nm.1-6 They have been opted over long-established semiconducting nanocrystals (QDs) and organic fluorophores owing to unique characteristics such as size and wavelength-dependent photoluminescence (PL), better photostability, greater aqueous solubility, lesser cytotoxicity and environmental amiability for bioimaging applications.7-11 In C-dots, carbon exists as a nanocrystalline graphitic core enriched with sp2-hybridized C-C linkages whereas the presence of carboxyl and hydroxyl functionalities on its surface account for its hydrophilicity.12,13 These benefits have been extensively employed to develop optically active sensors to detect biomolecules, intracellular organelles and noxious free radicals.14 Previously, several strategies such as surface passivation, functionalization, heteroatom doping were devised to expand their optical and biological applications.5,15,16 Among them, heteroatom doping has garnered more attention due to its facile synthesis procedures.4,10 After their chanced discovery, systematic investigations related to synthetic approaches and their applications were designed owing to their intrinsic traits. Nevertheless, these approaches had certain limitations such as necessity for costly and toxic reagents, intricate experimental


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requirements, multi-step procedures, treatment with acids/bases/oxidants and prolonged reaction time, which were far deviating from the idea of green chemistry.2,17 Thus, the development of highly fluorescent C-dots from naturally abundant and inexpensive raw materials emerged to be a significant task. Incidentally, bottom-up strategies such as pyrolysis,16,18 silica-supported synthesis,19 solvo/hydrothermal carbonization of carbohydrates derived from synthetic precursors2,20 or from naturally available precursors such as pomelo-peel, willow tree barks, egg shells and orange juice have been demonstrated.2,3,21-23 In this regard, microwave (MW)-assisted pyrolysis has emerged as the most feasible approach owing to efficient energy consumption, sustainability, lower temperature and shorter reaction time along with higher product yield.12,16,21 Regarding the synthesis of heteroatom-doped C-dots, the first synthesis was successfully reported by Sun et al. using Zn2+ ions (ZnO and ZnS) as precursors.24 Qian et al. successfully synthesized Si-doped C-dots from hydroquinone using silicon tetrachloride for bioimaging applications.25 Recently, non-metals like nitrogen, sulfur and boron were used as dopants to enhance the biocompatibility of C-dots.7 N-doped C-dots were extensively developed from synthetically derived precursors as well as from natural resources such as milk, grass, bee pollens and cocoon silk.26-31 Xu et al. successfully demonstrated the synthesis of sulfur-doped C-dots (S/C-dots) with 67% PL quantum yield (QY) using sodium citrate and sodium thiosulphate as precursors for detection of metal ions.32 Recently, phosphorus-doped C-dots were successfully developed for bio-labeling and catalytic oxygen reduction applications.33,34 Dong et al. devised a facile, single-step hydrothermal method to synthesize nitrogen and sulfur co-doped C-dots using citric acid and L-cysteine as precursors.35 Bourlinos et al. synthesized boron-doped C-dots (B/Cdots) to investigate on non-linear optical properties.36


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So far, most of the studies were mainly conducted on developing C-dots doped with nonmetals. However, studies regarding synthesis and applications of heavy metal-doped C-dots were rarely reported. Recently, Yang Xu and his co-workers synthesized gadolinium-doped C-dots (Gd-CQDs) as dual fluorescent and MRI probes.37 Quan Xu et al. developed Cu-doped C-dots as fluorescent sensing probes to detect Fe3+ ions.38 Recently, our group successfully developed Ag/C-dot and Au/C-dot nanohybrids from lemon extract for cancer cellular imaging.39 Several novel nanostructured hybrid materials have been successfully developed in our research laboratory using MW-assisted methods.40-45 Of late, “magneto-fluorescent” dual-modality contrast agents have emerged as efficient and successful probes in biomedical research.37,46 Primarily, metal oxide nanoparticles such as iron oxide nanoparticles (Fe2O3/Fe3O4), MnO, Gd2O3, Dy2O3 were utilized.47-50 However, these nanoparticles had shortcomings such as rapid renal disposal (Fe2O3/Fe3O4), high cytotoxicity (Gd2O3), feeble biocompatibility at lesser exposure time, reduced functionalization, lesser payload of magnetic centers and chelate etching.46 In addition, transition metals incorporated conjugated polymeric nanoparticles such as gadolinium-conjugated folate-polyethylene glycolpoly (amidoamine) dendrimer, magneto-fluorescent semiconducting polymer nanoparticles (MFSPNs) such as PPE, PFPV, F8BT and MEH-PPV conjugated with super paramagnetic iron oxide nanoparticles have also been reported as magneto-fluorescent dual-modality contrast agents.51,52 However, these contrast agents have large hydrodynamic size ranging from 50-700 nm, tedious and expensive synthetic strategies along with systemic toxicity at higher concentrations.52-54 Significantly, these challenges can be suitably overcome by viable combination of magnetic nanoparticles with enhanced MR efficacy and highly photoluminescent non-toxic quantum dots (QDs) using facile synthetic approaches. Although a few nanohybrids such as Gd-CQDs and iron


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oxide doped carbogenic nanocomposites (IO-CNC) have been successfully synthesized and implemented, the development of transition metal ion-doped carbonaceous QDs for magnetofluorescent bioimaging are still unexplored.37,55 Herein, we demonstrate a rapid synthesis of transition metal ion (Mn2+, Fe2+, Co2+, Ni2+)-doped C-dots (TMCDs) using lemon extract using one-pot, energy-efficient microwave-assisted pyrolysis method at 800 W within 6 min. The chief constituents of lemon extract namely Lascorbic acid (64 %), citric acid (6 %), carbohydrates (2.5 %) and trace amounts of flavonoid derivatives serve as carbon precursors to prepare TMCDs with better physico-chemical and optical properties.56 Interestingly, the EDA-functionalization on TMCDs enabled elimination of surface emissive traps to enhance the fluorescence emission and PL lifetime.16 Thus, the synthesized water-soluble TMCDs could serve as promising nanoprobes for fluorescent and T1magnetic resonance dual-modality bioimaging applications.

EXPERIMENTAL SECTION Materials. Fresh lemons were procured from a local vegetable market. Ethylenediamine (EDA) and Manganese acetate (Mn(OAc)2.4H2O) were purchased from Merck. Cobalt acetate (Co(OAc)2.2H2O) and Nickel acetate (Ni(OAc)2.2H2O) were purchased from Loba Chemie. Ammonium ferrous sulphate ((NH4)2Fe(SO4)2.6H2O) was purchased from Fischer Scientific Co. Dulbecco modified Eagle medium (DMEM), Fetal Bovine Serum (FBS), phosphate buffer saline (PBS) and MTT (3-(4,5-dimethylthiazol-2-yl)–2,5-diphenyltetrazolium bromide), penicillin and streptomycin to prepare 1% antibiotic solution were all purchased from Himedia. Dimethyl sulfoxide (DMSO) was purchased from Merck Biosciences. All the materials obtained were used without any further purification.


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Synthesis of EDA-functionalized C-dots using Citrus limon extract. In brief, 50 mL of fresh lemon juice was filter extracted and 9 mM (603 µL) EDA was added. The lemon extract/EDA mixture was irradiated in microwave oven at 800 W for 6 min. The solution color turned from turbid white to deep brown flakes indicating the pyrolytic carbonization of lemon extract to form EDA-functionalized C-dots. Synthesis of EDA-functionalized Transition Metal ion-doped C-dots (TMCDs). Briefly, 20 mL of lemon extract, 30 mL of 0.5 mM transition metal ion precursor and 9 mM (603 µL) EDA were mixed. Accordingly, 3.675 mg Mn(OAc)2.4H2O, 5.882 mg ((NH4)2Fe(SO4)2.6H2O), 3.735 mg Co(OAc)2.2H2O, 3.732 mg Ni(OAc)2.2H2O were used as transition metal ion precursors for Mn2+, Fe2+, Co2+, Ni2+ doping respectively. The solution mixture was irradiated in microwave oven at 800 W for 6 min. The solution color changed from turbid white to deep brown flakes, indicating simultaneous carbonization besides doping of transition metal ions onto the surface of EDA-functionalized C-dots to obtain TMCDs. The synthesized EDA-functionalized TMCDs powder was dissolved in water to form a solution. The resultant solution was then centrifuged at 10,000 rpm for 10 mins to precipitate the non-fluorescent aggregates and clear supernatant was used for analytical characterizations. For biological experiments, the supernatant solution was filter sterilized with 0.22 µm syringe filter. The schematic illustration of MW-assisted pyrolytic synthesis and characterization of EDA-functionalized TMCDs are shown (Scheme. 1). Material Characterization and Instruments. X-ray diffraction (XRD) of the synthesized EDA-functionalized C-dots and TMCDs was carried out by using Rigaku Ultima-IV X-ray diffractometer with Cu Kα radiation (λ = 1.54 Å), producing an acceleration voltage of 40 kV and 30 mA to scan the diffraction angles from 10-80°. High Resolution Transmission Electron Microscopy (HRTEM) images were attained using JEOL/JEM 2100 microscope in which


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lanthanum hexaborate (LaB6) was used as electron source at an accelerating voltage of 200 kV and magnification of 2000-1500000 X. The chemical structure and functional groups on TMCD surface were confirmed using Thermo Nicolet-6700 Fourier Transform Infrared (FTIR) spectrometer. To understand the semi-quantitative elemental composition of TMCDs, X-ray Photoelectron Spectroscopy (XPS) was carried out using Kratos Analytical Ltd X-ray photoelectron spectrometers. Additionally, Energy Dispersive X-ray (EDAX) analysis was also carried out using Carl-Zeiss Supra – 55 analyzer. The Zeta (ζ) potential of the as-prepared TMCDs was evaluated using HORIBA SZ-100 Analyzer. The absorbance spectra were measured using UV-3600 plus spectrophotometer (Shimadzu). The steady-state PL emission spectra were recorded using FLUOROLOG – FL3- 11 (Jobin Yvon) with excitation wavelengths from 300 to 480 nm and spectral resolution of 3 nm. Time-resolved photoluminescence (TR-PL) spectra were also measured using FLUOROLOG – FL3- 11 (Jobin Yvon) with 488 nm femtosecond laser. Vibrating Sample Magnetometry (VSM) analysis was performed to study the magnetic behavior of the synthesized TMCDs using Lakeshore VSM 7410. Quantum yield (QY) and PL Lifetime measurement. The PL QY of EDA-functionalized Cdots and TMCDs was calculated using Rhodamine-6G (dissolved in ethanol, QY = 0.95) as reference by the equation:28,39,40 Φs = Φr (Fs/Fr) *(Ar/As)*(n2s/n2r) -------- (1) where Φ represents quantum yield, F denotes integrated PL emission intensity, A denotes absorbance at the maximum excitation wavelength and n is the refractive index of the solvent (n = 1.33 for water, n = 1.36 for ethanol). The subscripts s and r represent the sample and reference solutions respectively. The amplitude-weighted average lifetime (τav) was calculated using the equation:28,56,57


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τav = B1τ12 + B2τ22 + B3τ32 / B1τ1 + B2τ2 + B3τ3 -------- (2) where B1, B2 and B3 denote the relative amplitudes of the decay factors with respective decay times τ1, τ2 and τ3. In vitro cytotoxicity studies. To estimate the cellular viability of EDA-functionalized TMCDs, MTT Assay was performed.58 Cancerous cell lines SW480 (human colon adenocarcinoma) was purchased from National Centre for Cell Science (NCCS), Pune, India. SW480 cells were cultured in 96-well flat-bottomed culture plate at a density of 5 x 103 cells/well in 100 µL of DMEM supplemented with 10 % FBS for 24 h. After incubation, the cells were treated with different concentrations of the EDA-functionalized C-dots and TMCDs (31.25, 62.5, 125, 250, 500, 1000, 2000 µg/mL respectively) for 24 h and 48 h. The culture medium was removed and 20 µL of MTT solution (5 mg/mL concentration) was added. The plate was incubated at 4 h at 37 ⁰C in the dark to form purple-colored formazan crystals which were dissolved in 100 µL of DMSO. The formazan metabolite was quantitatively measured as a function of optical density (OD) value obtained at 570 nm. The OD values were measured using ELISA plate spectrophotometer and the cell-viability was calculated using the equation:28,58 Cell viability (%) = (As/Ac) x 100 -------- (3) where ‘A’ represents optical density (OD). The subscripts s and c denote sample and control respectively. In vitro Fluorescence Imaging. The SW480 cells were seeded in a 6-well plate at a density of 105 cells/well and incubated in a humidified 5 % CO2 incubator at 37 ⁰C. After 24 h, cells were treated with fresh culture media containing 100 µL of 100 µg/mL concentration of EDAfunctionalized TMCDs and incubated for 4 h. Cells were then washed twice with PBS (pH 7.2)


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and fixed with 4 % paraformaldehyde (PFA) for 15 mins. Cells were observed under confocal microscope (LSM700, Carl Zeiss) for capturing the localization of TMCDs. Zebrafish husbandry. Wild-type zebrafish were maintained under standard conditions at 28.5ºC with 10/14-h dark/light cycle.40 They were fed with freshly hatched live brine shrimp and formulated pellets. All experimental protocols involving zebrafish were approved by the Institutional Animal Ethics Committee (IAEC) of SRM Institute of Science and Technology, Kattankulathur, India. All the methods were carried out strictly in accordance with the approved guidelines. Radiant efficiency and in vivo fluorescence imaging. The total radiant efficiency of EDAfunctionalized TMCDs was measured by using agarose phantom. Different concentration of transition metal ions in TMCDs ranging from 1 mM – 5 mM were suspended in agarose gel (1%, w/v) in a 48-well plate. For in vivo imaging, the zebrafish were anesthetized with tricaine (3aminobenzoic acid ethyl ester) as per standard protocol and subsequently rapidly injected with 10 µL of 5 mg/mL EDA-functionalized TMCDs interperitonially by microloader pipette. Fluorescence imaging was performed using IVIS Lumina LT Series III, PerkinElmer, USA. The fluorescence emitted at 460 nm was imaged using Peltier cooled CCD camera (-90 ˚C) to ensure low dark current and low noise after exciting at 400 nm. All the images were uniformly scaled and analyzed by using Living Image Software version 4.3.1. Relaxometric studies and in vivo MRI. The MRI relaxivities of EDA-functionalized TMCDs was measured by using agarose phantom. Different concentration of transition metal ions in TMCDs ranging from 1 mM – 5 mM were suspended in agarose gel (1%, w/v) in a 48-well plate. For in vivo imaging, the zebrafish were anesthetized with tricaine (3-amino benzoic acidethylester) as per standard protocol and subsequently rapidly injected with 10 µL of 5


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mg/mL EDA-functionalized TMCDs interperitonially by microloader pipette. The relative relaxivities (r1 and r2) were determined by measuring relaxation times (T1 and T2) with a 1.5 T (Philips Achieva)-MRI scanner using radio frequency (RF) coil to validate the efficacy of EDAfunctionalized TMCDs as MRI contrast agents. To carry out relaxometric measurements, T1weighted images were obtained using the following fixed parameters – Field of view = 120 x 120 mm, repetition time (TR) of 450 ms and echo time (TE) of 15 ms, flip angle of 69° and 2 signal acquisitions. A spin echo (SE) T1-weighed imaging with repetition time of 460 ms, echo time of 30 ms, No. of excitations (NEX) = 1, Slice thickness (S.T) = 1 mm, Imaging matrix = 256 x 192 mm, Field of view (F.O.V) = 80 x 80 mm. Similarly, T2-weighted images were obtained using Field of view = 120 x 120 mm, TR of 2921 ms and TE of 100 ms and 2 signal acquisitions. The signal intensity on magnitude images was averaged within regions of interest (ROI) and echo time was plotted for T1 and T2 decay curves for all the samples. The data were fitted to the mono-exponential function:46 T (1,2) = A (1-e-TR/T1,2) --------(4) The relaxation rates R1 and R2 were obtained by reciprocal of the relaxation times (T1 and T2), and were plotted against different concentration of TMCDs and relaxivities (r1 and r2) were calculated as slope of the resultant tangent plot by using the equation below46,55 R1,2 = r1,2 [C] + R1,2* -------- (5) where R1,2* is the relaxation rate of control and [C] is the transition metal ion precursor concentration in mM. RESULTS AND DISCUSSIONS The stages involved during the synthesis of EDA-functionalized pristine C-dots and TMCDs from lemon juice comprise of a facile pyrolytic carbonization of its constituents together with


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doping of transition metal ions (Mn2+, Fe2+, Co2+, Ni2+). During MW-assisted pyrolysis, the microwave radiation falls upon the molecules of precursor solution, leading to immediate and localized heat transfer. For pristine C-dots, lemon extract undergoes dehydration coupled with decomposition of biomolecules to form intermediate products such as furfural derivatives, organic acids, aldehydes and phenols. The hydronium ions present in the intermediary organic acids catalyze the ensuing decomposition stages. The furfural derivatives and organic acids undergo further polymerization and condensation to form soluble polymers, which aromatizes via aldol condensation, cycloaddition and hydroxymethyl-mediated furan resin condensation to form aromatic clusters. These aromatic clusters get concentrated and attain a critical supersaturation point at which they undergo nucleation burst to form the resultant carbon dots.2,23 However, in TMCDs the metal precursors on direct interaction with microwave radiation do not get reduced to atomic or metallic species. Instead, these precursor salts get ionized and physically interact with the oxygen atoms of the oxo-functional groups on C-dot surface. Furthermore, the MW-assisted pyrolysis was carried out at 800 W (at nearly 150 °C) under ambient conditions, subsequently favoring the formation of corresponding metal oxide traces on C-dot surface. Interestingly during MW-assisted pyrolysis, EDA gets protonated in aqueous medium and couples with carbon precursors to form surface amide linkages that effectively mask the surface-emissive trap sites resulting in enhanced PL and simultaneously serves as surfacecapping agent to form EDA-functionalized TMCDs.16,61 Analytical Characterization. X-ray diffraction peaks of the EDA-functionalized pristine C-dots and TMCDs are shown (Figure 1). A broad amorphous (002) peak was observed at 19.18° for pristine C-dots and was consistent with previous report.23 The synthesized Mn/C-dots, Fe/C-dots, Co/C-dots and Ni/C-dots exhibited a minor shift to 17.94, 19.14, 18 and 18.52° respectively. The


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corresponding d-value for the synthesized pristine C-dots was calculated using Scherrer’s formula and found to be 0.26 nm. However, the d-values for TMCDs were found to be 0.33 (Fe/C-dots), 0.35 (Mn/C and Co/C-dots), 0.36 nm (Ni/C-dots), which were close to that of graphite (0.34 nm).23 The size and morphology of the EDA-functionalized TMCDs were examined using HRTEM images as presented (Figure 2). The TMCDs were homogeneously dispersed and exhibited nearly spherical morphologies. The particle sizes of Mn/C-dots, Fe/C-dots, Co/C-dots and Ni/Cdots were measured to be 3.12, 2.73, 3.69 and 2.72 nm respectively. Also, the selected area electron diffraction (SAED) patterns and particle size distributions for all the TMCDs are shown in the insets (Figure 2). The interlayer spacing of TMCDs was found to be 0.35 ± 0.01 nm which was exactly corresponding to (002) plane of sp2-graphitic carbon and closely matching with dspacing values attained from XRD analysis.23 The chemical composition and major functional groups present on the surface of EDAfunctionalized pristine C-dots and TMCDs were examined using FTIR spectroscopy (Figure 3). Significantly, the vibrational C=C bands were formed at 1641 cm-1 for pristine C-dots, 1654 cm-1 for Mn/C, Fe/C and Co/C-dots and 1658 cm-1 for Ni/C-dots which confirmed the occurrence of sp2-graphitic C-atoms. A sharp C=O stretching vibration peak was observed at 1709 cm-1 for pristine C-dots, 1707 cm-1 for both Mn/C and Fe/C-dots, 1700 and 1702 cm-1 for Co/C and Ni/Cdots respectively, indicating the occurrence of aromatic carbonyl linkages on C-dot surface. Similarly, a faint peak was noticed at 1778 cm-1 for pristine C-dots and Ni/C-dots, 1774 cm-1 for Mn/C and Fe/C-dots and 1832 cm-1 for Co/C-dots due to stretching C=O vibrations, indicating the formation of weak anhydride linkages on C-dot surface. Furthermore, the EDAfunctionalized C-dots and TMCDs revealed C-O-H stretching at 1400 cm-1 due to the presence of


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sp2-carbon atoms on C-dot surface. Also, aliphatic C-O stretching was observed at 1296 and 1186 cm-1, 1295 and 1124 cm-1, 1300 and 1087 cm-1, 1296 and 1189 cm-1 for Mn/C, Fe/C, Co/C and Ni/C-dots respectively. Due to EDA-functionalization, aliphatic C-O stretching was revealed at the peaks 1172, 1082 and 900 cm-1. The synthesized pristine C-dots and TMCDs revealed asymmetric C-H (methyl) stretching vibrations at 2950, 2923, 2927, 2983 and 2952 cm-1. A broad hydroxyl (-OH) peak was attained at 3372, 3448, 3396 and 3361 cm-1 for the synthesized TMCDs due to stretching vibrations of -OH moieties. A faint peak was found at 3099 cm-1 for pristine C-dots, at 3097 cm-1 for both Mn/C and Ni/C-dots, at 3095 and 3110 cm-1 for Fe/C and Co/C-dots respectively due to -NH2 stretching vibrations. A peak at 1560 cm-1 for both Mn/C and Fe/C-dots, 1577 cm-1 for Co/C-dots and 1562 cm-1 for Ni/C-dots appeared owing to deformed -NH2 vibrations which confirmed EDA-functionalization on C-dot surface. Interestingly, the spectra of all TMCDs indicated several minute peaks characteristic of metal oxides below 1000 cm-1 which confirmed the occurrence of metal oxide traces on TMCD surface. Specifically, the spectra of Mn/C-dots denoted very tiny peaks at 484, 520 and 617 cm-1 due to stretching vibrations of Mn-O bonds of Mn2O3. Also, a miniscule peak was noticed at 793 cm-1 owing to bending vibrations of O-C-O bonds coupled with O-Mn-O bridge.62 In the spectra of Fe/C-dots, a miniscule peak at 455 cm-1 and a medium peak at 619 cm-1 were observed due to stretching and torsional vibration modes characteristic of Fe-O bonds corresponding to haematite (Fe3O4) traces at tetrahedral and octahedral sites respectively.63 Co/C-dots also yielded feeble peaks at 582 and 667 cm-1 which were ascribed to Co-O stretching, confirming the presence of CoO traces.64 Similarly, Ni/C-dots revealed a feeble peek at 424 cm-1 due to strong bands caused by Ni-O variations, thus confirming the presence of trace amounts of NiO on Ni/C-dot surface.65 The signature peaks of the synthesized TMCDs has been listed (Table 1).


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To understand the surface composition and electronic states of the constituent elements present in the EDA-functionalized TMCDs, XPS analysis was carried out (Figure 4). In common, the high-resolution survey scan of the TMCDs revealed three peaks of C (1s), N (1s) and O (1s) at the range of 285.5 ± 0.2, 399.5 ± 0.2 and 530.0 ± 0.3 eV respectively. The C (1s) spectrum of the TMCDs further splits into three peaks at binding Energies (B.Es) of 284.5 ± 0.2, 285.9 ± 0.2 and 288.1 ± 0.2 eV which are attributed to C=C, C-N and C=O/C=N respectively.3,14 However, Co/C-dots revealed two additional peaks at 287.2 and 289.2 eV which are assigned to C-O and O-C=O bonds respectively.10,66 The high-resolution spectrum of N (1s) of all the TMCDs revealed a dominant peak at 400 ± 0.1 eV which is attributed to N-(C)3.10,27 In addition, a medium peak was noticed at 401.8 eV for both Mn/C-dots and Fe/C-dots and at 401.3 eV for Co/C which are attributed to N-H bonds.10,27 Mn/C-dots and Co/C-dots revealed minor peaks at 398.2 and 399.1 eV respectively due to the presence of pyridinic C-N-C and pyrrolic N-(C)3 respectively.27 The deconvolution of O (1s) spectrum yielded a major peak at 531.6 ± 0.3 eV due to C=O (carbonyl linkages).3,9 Also, Co/C-dots revealed an additional peak at 533.7 eV which was ascribed to C-OH/C-O-C group.3 In Mn/C-dots, the feeble Mn (2p) peak was resolved into three peaks namely Mn (2p)3/2 (1) and Mn (2p)3/2 (2) at B.Es of 641.1 and 642.9 eV respectively and Mn (2p)1/2 at 653.55 eV with a standard separation of 12.2 eV (Figure 4(a). The resolved Mn (2p)3/2 and Mn (2p)1/2 were assigned to Mn-O bonds of MnO and Mn2O3 respectively. The presence of satellite peak at 646.95 eV confirmed the presence of divalent and trivalent states of Mn.67-69 In Fe/C-dots, the feeble Fe (2p) peak was deconvoluted into two peaks of Fe (2p)3/2 and Fe (2p)1/2 at B.Es of 709.95 and 708.31 eV respectively (Figure 4(e). Further deconvolution of Fe (2p)3/2 peak reveals three peaks at B.Es of 708.3 and 710.1 eV which are both ascribed to the occurrence of Fe-O bonds of Fe2O3 and Fe3O4 traces on Fe/C-dot surface. Also, a medium peak


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at 713.3 eV which was attributed to multiplets of Fe3+ ions from Fe3O4 and γ-Fe2O3, indicating their co-existence.70-72 In Fe (2p)1/2spectrum, a medium peak at 724.4 eV was assigned due to FeO bonds of FeO.73 In Co/C-dots, the feeble Co (2p) peak further splits into two peaks (Figure 4 (i). A major peak of Co (2p)3/2 located at 780.25 eV was attributed to Co-O bonds of CoO traces.74 A shake-up satellite was observed at 785.07 eV, with a shift of 4.82 eV from Co (2p)3/2 peak due to charge-transfer band structure characteristic of 3d-transition metal oxide, thus conforming the chemical oxidation of cobalt in Co/C-dots as +2.45 In Ni/C-dots, the weak Ni (2p) peak was deconvoluted into three major peaks at 856.13, 861.11 and 864.8 eV (Figure 4 (m). The Ni (2p) core level spectrum specified the Ni atoms in (2p)3/2 which confirmed the divalent states of Ni atoms. The peak at 856.13 eV was ascribed to Ni (2p)3/2 due to Ni-O bonds of NiO traces, thus indicating the doping of Ni2+ ions with oxygen atoms of oxo-functional groups on Cdot surface, with a preferred oxidation state of +2.45 The occurrence of a satellite peak at 861.11 eV was ascribed to Ni (2p)3/2 due to binding of Ni2+ ions with hydroxyl moieties.75 The peak at 864.8 eV was assigned to Ni-O bonds between Ni2+ ions and oxygen atoms of carbonyl/carboxylic acid moieties on C-dot surfaces.76 Thus the surface amino and oxy functional groups as well as M-O bonds (where M denotes Mn2+/Fe2+/Co2+/Ni2+) confirmed the surface functionalization and metal ion doping in TMCDs. The occurrence of transition metal ions in EDA-functionalized TMCDs was further confirmed by EDAX analysis (Figure S2-Supporting Information). Carbon, oxygen and nitrogen constituted weight percentages of 51.34 ± 3.46, 31.84 ± 3.03 and 18.4 ± 3.9 % respectively. The obtained weight percentages of nitrogen and oxygen for the synthesized TMCDs were attributed to amine and oxy-functional groups formed on the C-dot surfaces and were consistent with the obtained FTIR and XPS results as well. The incorporation of transition metal ions onto C-dots was


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notably validated with weight percentages of 0.16 % of Mn in Mn/C, 0.56 % of Fe in Fe/C, 0.38 % of Co in Co/C and 0.13 % of Ni in Ni/C-dots. The lesser weight percentage of metals was attributed to the formation of corresponding metal oxide traces and very low precursor concentration (0.5 mM). The zeta potential (ζ) potential analyses of the non-functionalized and amine-functionalized TMCDs were carried out (Figure 5). All the C-dot samples exhibited negative ζ-potential in the following order: Non-functionalized C-dots (-39.8 mV) < Mn/C (-37.5 mV) < Co/C (-36.4 mV) < Fe/C (-28.9 mV) < Ni/C (-28.4 mV) < amine-functionalized C-dots (-25.2 mV). The non-functionalized pristine C-dots yielded a negative zeta (ζ) potential of -39.8 mV. However, EDA-functionalization resulted in doping of positively charged amine moieties on the pristine C-dots surface, thereby increased to -25.2 mV. On the contrary, the ζ-potential of the synthesized amine-functionalized TMCDs was increased to -33 ± 5 mV due to the presence of transition metal ions on the C-dot surface as evident from FTIR and XPS data. The negative charge for the C-dot samples was attributed to rich abundance of carboxylic/carbonyl and hydroxyl moieties on the surface, thus confirming their superb colloidal stability in the aqueous medium. The stability of EDA-functionalized TMCDs under physiological condition was studied in PBS buffer and drastic change in ζ potential was observed with respect to water. The TMCDs exhibited near neutral ζ potential over 24 h (Figure S3-Supporting Information). The PBS solution, which has high ionic strength contains strong cations (Na+, K+) as well as strong anions (PO42-, Cl-). When TMCDs are dissolved into PBS, the cations in PBS could form salt bridges with negative charged oxo-functionalities (-COOH, -OH) on the C-dots surface. Similarly, the anions could form salt bridges with positively charged amine functionalities (-NH2) of C-dots


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and transition metal cations (Mn2+, Fe2+, Co2+, Ni2+). The formation of these salt bridges could have resulted in masking the surface charges present on TMCDs and effectively neutralizing the overall ζ potential.77,78 However, reduced negative ζ potential for TMCDs in PBS could be attributed to the presence of excess negatively charged oxo-functionalities on the C-dots surface. The absorbance spectra of EDA-functionalized pristine C-dots and TMCDs were analyzed (Figure 6). With respect to pristine C-dots, Mn/C and Fe/C-dots exhibited increased absorbance whereas Co/C and Ni/C-dots presented decreased absorbance. For the synthesized samples, a broad characteristic peak of C-dots was observed at 350 ± 3 nm due to n-π* transition. Correspondingly, a dip was noticed at 285 ± 3 nm due to π-π* electronic transition owing to surface-bending characteristic of graphitic sp2-carbon on C-dots surfaces.12 The band-gap energy was calculated using Einstein-Planck relation E = hc/λ and was found to be 3.54 eV. The steady-state PL emission spectra of EDA-functionalized pristine C-dots and TMCDs (Mn/C, Fe/C, Co/C and Ni/C-dots) were recorded (Figure 7 (a-e). The synthesized pristine C-dots showed optimal excitation wavelength (λex) centered at 460 nm and corresponding bright PL emission at 517 nm with a PL QY of 48.31 %. The synthesized TMCDs exhibited excitation wavelength-dependent PL emission. This occurrence was due to the existence of two types of emissive traps besides core exciton recombinant pairs. Firstly, the oxo-functionalities which constitute the primary surface states present on the carbon core formed due to partial oxidation of precursors during pyrolytic synthesis of C-dots. Secondly, the amine functional groups present on C-dot surfaces formed due to surface functionalization caused by addition of ethylenediamine. These surface-emissive traps are associated with a definite amount of energy and get activated on excitation by a certain wavelength and therefore attribute to wavelengthdependent PL behavior.16


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In the case of TMCDs, the enhancement or quenching of PL depends upon the interaction of transition metal ions which act as dopants with the surface of C-dots. The PL quantum yield (PL QY) was calculated to be 35.71, 41.72, 75.07 and 50.84 % for Mn/C, Fe/C, Co/C and Ni/C-dots respectively. The decreased PL QY for Mn/C and Fe/C-dots were attributed to formation of complexes between Mn2+/Fe2+/Fe3+ ions with amine and hydroxyl functional groups on C-dot surfaces, leading to splitting of 3d-orbital of metal ions. This results in partial electron transfer from the excited state of EDA-functionalized C-dots to 3d-orbital of metal ions, leading to loss of radiative recombination and eventually resulting in fluorescence quenching.10,79-81 However, in Ni/C-dots, Ni2+ ions exhibit lesser absorption affinity to form complexes with C-dot surface functional groups with respect to their Mn and Fe counterparts, leading to lesser formation of complexes. This leads to reduced electronic transfer, thus generating a partial fluorescence quenching and slightly enhanced QY.82 In Co/C-dots as well, high Co2+ ion concentrations favors chelation between Co2+ ions and oxo-functionalities on C-dot surface resulting in the formation of Co2+-based complexes, particularly with hydroxyl (-OH) moieties to form insoluble Co(OH)2 which causes effective PL quenching. However, low Co2+ ion precursor concentration (0.5 mM) effectively prevents the formation of complexes and the secondary amine moieties present on C-dot surface emissive trap sites bind to primary oxy-functional groups resulting in high radiative exciton recombination and subsequent PL QY enhancement in Co/C-dots.83 The PL lifetime (τav) of EDA-functionalized pristine C-dots and TMCDs was measured using time-correlated single photon counting (TCSPC) analysis (Figure 7 (f). The decay curves were tri-exponentially fitted and the average PL decay (exciton lifetime) of pristine C-dots, Mn/C, Fe/C, Co/C and Ni/C-dots were calculated to be 3.6, 9.4, 8.6, 9.2 and 8.9 ns respectively. The shorter life-time for pristine C-dots was attributed to radiative recombination of carbon core


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excitons.28,56 Significantly, the enhanced exciton lifetime in TMCDs could be attributed to prolonged excitation localization between core excitons and carrier surface-associated radiative recombination. The radiative transition occurring between localized hole and electron quantized state results in slower radiative recombination and subsequent higher PL emission lifetime.40 VSM analysis of the synthesized pristine C-dots and TMCDs are presented (Figure 8). From the magnetic hysteresis (M-H) curves recorded at room temperature, the corresponding coercivity, magnetic saturation and retentivity values were determined (Table 2). The synthesized pristine C-dots exhibited diamagnetic behavior with negative magnetic saturation while TMCDs demonstrated positive magnetic saturation. The greater magnetization was observed in superparamagnetic Ni/C-dots (28.11 m emu/g), followed by ferromagnetic Co/C-dots (15.59), Mn/C-dots (14.78) and Fe/C-dots (9.95 m emu/g), which confirmed their potential as T1weighted contrast agents for magneto-fluorescent bioimaging applications. Biological Characterizations In vitro Cytotoxicity Studies. To demonstrate TMCDs as magneto-fluorescent nanoprobes for bioimaging application, QDs should possess certain essential inherent characteristics such as colloidal stability, enhanced fluorescence and high biocompatibility.4 The extent of cytotoxicity of EDA-functionalized C-dots and TMCNs were evaluated using MTT assay on SW480 cells (human colon adenocarcinoma) cells. Interestingly, the cells exhibited no cytotoxicity even at a higher concentration of 2 mg/mL for 24 hrs and 48 hrs (Figure 9 a,b) respectively). A higher cell viability was previously reported using synthetic precursors such as PEG at an exceptionally high concentration of 10 mg/mL.84 Nevertheless, a high level of cell viability at higher concentration (2 mg/mL) of transition metal ion-doped C-dots derived from lemon extracts has


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never been reported. Recently, we had reported cent percent viability for Ag/Au-carbon nanohybrids at 1 mg/mL concentration prepared using lemon extract.39 In vitro fluorescence uptake studies. The potential of EDA-functionalized TMCDs as fluorescent probes has been illustrated by assessing their intracellular uptake (Figure 10). A distinct cellular morphology and bright green fluorescence was clearly observed indicating facile internalization of TMCDs into the cell. Significantly, the green fluorescence was also observed in the nucleus within 4 h highlighting the entry of TMCDs into the nucleus due to its smaller size (< 9 nm) through nuclear pore complexes.66 Radiant efficiency and in vivo fluorescence imaging. The bioimaging potential of EDAfunctionalized TMCDs were confirmed in agar and Zebrafish using IVIS Lumina LT Series III, PerkinElmer. The TMCDs suspended in agar phantom showed bright fluorescence with increasing transition metal ion concentration in 48-well plate (Figure 11 (a). The fluorescence intensity in the regions of interest (ROI) was measured to determine total radiant efficiency (Figure 11 (c).85 Fe/C-dots presented maximum radiant efficiency followed by Ni/C-dots, Mn/Cdots and Co/C-dots in the agar gel. Zebrafish model was further used to confirm the fluorescence response of TMCDs in vivo (Figure 11 (d). Zebrafish is one of the rapidly emerging model because of its flexibility to genetic manipulation and optical transparency (Figure 11 (c). After interperitonial injection of 10 µL EDA-functionalized TMCDs, zebrafish exhibited bright detectable fluorescence with greater signal to noise ratio and superior total radiant efficiency. Relaxometric studies and in vivo MRI. The potential ability of EDA-functionalized TMCDs as effective T1 and T2-weighted MR-contrast agents were confirmed by performing relaxometric studies in agar phantom and Zebrafish using 1.5 T (Philips Achieva)-MRI scanner (Figure 12 (a, b). From the longitudinal (r1) and transverse (r2) relaxivity graph, the Mn/C-dots, Fe/C-dots,


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Co/C-dots showed an apparent decreasing relaxation with increasing concentration of transition metal ions owing to higher relaxation time (Figure 12 (c, d). However, Ni/C-dots showed enhancement of relaxation with increase in Ni2+ concentration which confirmed the superparamagnetic behaviour as evident from VSM. The Mn/C-dots exhibited maximum longitudinal (r1) and transverse (r2) relaxivities of 0.341 and 2.015 mM-1s-1 which were found to be comparable with clinically used Gd-DTPA MR-contrast agents (longitudinal relaxivity of 3.13.5 mM-1s-1).86 To determine the efficiency of MR contrast agents, relaxivity ratio (r2/r1) is a major parameter. The substance normally behaves as T1-contrast agent if the relaxivity ratio (r2/r1) is < 2 and as T2-contrast agent if r2/r1 is ≥ 10.51 The relaxivity ratios for EDAfunctionalized TMCDs were calculated for increasing concentrations transition metal ions (Table S1-Supporting information). From the relaxivity studies, it was found that the synthesized Mn/C-dots, Fe/C-dots, Ni-C-dots could serve as predominant T1-weighted MRI contrast agents and Co/C-dots which could serve as effective T2-weighted MRI contrast agents. The reduced relaxivity ratio could be due to incompatibility of the carbogenic core which effectively masks the magnetic configuration of transition metal ions. The magnitude of r1 could be attributed to reduced magnetic moment and lesser extent of dipole interaction between metal ion core and protons of water molecules. Conversely, the magnitude of r2 could be attributed to miniscule ionic size within the C-dots, heterogeneous magnetic field around peripheral water molecules and magnetic anisotropy factors arising due to morphology and surface effects.51 The in vivo relaxivity studies carried out in zebrafish showed distinct contrast detectable at the site of injection in both T1 and T2 images (Figure 12 (b). The apparent concentration of transition metal ions in zebrafish host were determined by extrapolating the linear fitting of relative relaxation rate values against the transition metal ion concentration (Table S2-Supporting information).


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The results indicated that the total radiant efficiency via optical imaging was significant even at lower concentration of transition metal ions. Conversely, MR imaging presented higher distribution of transition metal ions concentration for T1-weighted contrast ability. The MR response indicated depth penetration and special resolution of the TMCDs. Comparison between fluorescence and MR imaging results confirmed that EDA-functionalized TMCDs were implanted in the zebrafish and their magneto-fluorescent efficacy were at detectable levels. Thus, the facile preparation, ease of functionalization, high stability and low toxicity of lemon extractderived TMCDs could further promote the development of enhanced fluorescence and T1weighted MR-contrast agents for dual modality bioimaging applications. CONCLUSION In summary, one-pot microwave-assisted pyrolytic “green” syntheses of EDA-functionalized TMCDs have been successfully demonstrated within 6 mins using lemon extract. XRD peaks of TMCDs revealed amorphous and disordered graphitic structure with marginal shifting due to incorporation of metal ions on C-dot surface. The EDA-functionalized TMCDs revealed a spherical morphology with average particle size of 3.2 ± 0.485 nm and interlayer spacings of 0.35 ± 0.01 nm which were closely matching with that of sp2-graphitic carbon. The chemical composition and surface functional groups of the TMCD samples were undeniably confirmed by FTIR and XPS analyses. Also, the presence of dopant metal ions in trace amounts within the Cdots was confirmed by EDX analysis. The EDA-functionalized TMCDs together with nonfunctionalized pristine C-dots and amine-functionalized C-dots revealed negative zeta (ζ) potential due to rich abundance of oxo-functional groups which accounted for their better colloidal stability in aqueous environment. Both pristine C-dots and TMCDs revealed characteristic absorbance peaks due to π-π* electronic transition, excitation wavelength-


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dependent PL behavior, significant PL-QY and dynamic lifetime. The decreased PL emission intensity and resultant QY for Mn/C-dots and Fe/C-dots were attributed to non-radiative electronic transition caused by formation of complexes between Mn2+/Fe2+ ions and C-dot surface functional groups. However, enhanced PL emission and QY were observed for Co/Cdots due to inhibition of co-ordination complex formations between Co2+ ions and C-dot surface functional groups, caused by synergistic effect of reduced absorbance, very low Co2+ ion concentration and the binding between primary oxo-functional group and secondary amine group. Interestingly, prolonged exciton localization led to slower radiative recombination and resulted in enhanced PL lifetime for TMCDs (8.6 – 9.4 ns) with respect to pristine EDAfunctionalized C-dots (3.6 ns). The EDA-functionalized TMCDs except Ni/C-dots showed ferromagnetic saturation behavior. The MTT assay exhibited high biocompatibility and no cytotoxicity even at a higher concentration of 2 mg/mL. Intracellular fluorescence uptake images revealed the facile entry of TMCDs via cell membrane into the cytoplasm and partial diffusion into the nucleus due to their smaller size (< 9 nm). The agar phantom, in vivo fluorescence and magnetic resonance imaging confirmed EDA-functionalized TMCDs as suitable agent for T1weighted contrast with bright green emission at the administrable quantities. Thus, TMCDs prepared via microwave-assisted pyrolytic method could offer a promising magneto-fluorescent nanoprobe for dual-modality bioimaging.


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Scheme 1. Schematic illustration of microwave-assisted pyrolytic synthesis of EDAfunctionalized TMCDs as potential nanoprobes for magneto-fluorescent bioimaging.


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Figure 1. XRD peaks of EDA functionalized (a) pristine C-dots, (b) Mn/C-dots, (c) Fe/C-dots, (d) Co/C-dots and (e) Ni/C-dots.


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Figure 2. HRTEM images of EDA-functionalized TMCDs (a) Mn/C-dots, (b) Fe/C-dots, (c) Co/C-dots and (d) Ni/C-dots. Insets denote SAED pattern, particle size distribution and their interlayer spacings.


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Figure 3. FTIR spectra of EDA-functionalized (a) pristine C-dots, (b) Mn/C-dots, (c) Fe/C-dots, (d) Co/C-dots and (e) Ni/C-dots.


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Figure 4. High-resolution XPS spectra of EDA-functionalized TMCDs (a-d) Mn/C-dots, (e-h) Fe/C-dots, (i-l) Co/C-dots and (m-p) Ni/C-dots.


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Figure 5. Zeta (ζ) potential of (a) non-functionalized pristine C-dots, EDA-functionalized (b) pristine C-dots, (c) Mn/C-dots, (d) Fe/C-dots, (e) Co/C-dots and (f) Ni/C-dots dissolved in water.


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Figure 6. UV-Visible Spectroscopy of EDA-functionalized pristine C-dots and TMCDs.


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Figure 7. Steady state PL spectra of EDA-functionalized (a) pristine C-dots, (b) Mn/C-dots, (c) Fe/C-dots, (d) Co/C-dots and (e) Ni/C-dots and (f) time-resolved PL spectra of pristine C-dots and TMCDs.


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Figure 8. VSM analysis of the synthesized EDA-functionalized pristine C-dots and TMCDs.


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Figure 9. Cell viability studies using MTT Assay for the synthesized EDA-functionalized TMCDs at (a) 24 hrs and (b) 48 hrs with human colon adenocarcinoma (SW480) cells.


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Figure 10. Intracellular fluorescence uptake images of human colon adenocarcinoma (SW480) cells treated with EDA-functionalized TMCDs.


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Figure 11. (a) Agar phantom dispersed with increasing transition metal ion concentration of EDA-functionalized TMCDs in 48-well plate under in vivo imaging system. (b) Total radiant efficiency of EDA-functionalized TMCDs dispersed in agar phantoms plotted against increasing concentration of transition metal ion. The scatter points refers to total radiant efficiency of zebrafish extrapolated against transition metal ion concentration (c) Illustration of the zebrafish husbandry and microinjection of EDA-functionalized TMCDs. (d) In vivo fluorescence imaging of zebrafish injected with 10 µl of EDA-functionalized TMCDs under in vivo imaging system.


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Figure 12. (a) Agar phantom dispersed with increasing transition metal ion concentration of EDA-functionalized TMCDs in 48-well plate, (b) T1-weighted (above) and T2-weighted (below) MR contrast images of zebrafish injected with 10 µl of EDA-functionalized TMCDs viewed under 1.5 T (Philips Achieva)-MRI scanner. Circle denotes the site of TMCDs administration in zebrafish (c) Longitudinal relaxivity (r1) and transverse relaxivity (r2) graphs of EDAfunctionalized TMCDs dispersed in agar phantoms plotted against increasing transition metal ion concentration. The dotted lines refer to the relaxation rate of zebrafish extrapolated against transition metal ion concentration.


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Table 1. FT-IR analysis of synthesized EDA-functionalized pristine C-dots and TMCDs. Functional groups and Wave Number (cm-1)

Sample C=C

C=O

C-O

C-H

N-H

O-H

M*-O

Pristine C-dots

1650

1709

1080 – 1400

2950

1560

3415

-

Mn/C-dots

1654

1707

1180 - 1402

2923

1560

3372

480 - 620

Fe/C-dots

1654

1707

1120 - 1402

2927

1560

3448

455 - 770

Co/C-dots

1654

1700

1080 - 1410

2983

1577

3396

580 - 670

1657 1702 1180 - 1402 2952 1562 Ni/C-dots M* denotes Mn, Fe, Co, Ni in their appropriate metal-doped C-dots.

3361

424

Table 2. Magnetic properties such as coercivity (G), magnetization and retentivity (emu/g) of the synthesized EDA-functionalized pristine C-dots and TMCDs. Sample

Coercivity (G) 1189.9

Magnetization (emu/g) - 9.02 x 10-3

Retentivity (emu/g) 0.37 x 10-3

Mn/C-dots

352.52

14.78 x 10-3

0.33 x 10-3

Fe/C-dots

489.89

9.95 x 10-3

0.47 x 10-3

Co/C-dots

422.95

15.59 x 10-3

0.3 x 10-3

Ni/C-dots

22.55

28.11 x 10-3

0.46 x 10-3

C-dots

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge in ACS Publication Website on DOI: Supporting information contains a brief description on concentration optimization of ethylenediamine, Figure S1 represents steady-state PL spectra of C-dots with optimized


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ethylenediamine concentrations, Figure S2 represents EDAX and Figure S3 represents Zeta potential of EDA-functionalized C-dots in phosphate buffer saline. Table S1 representing relaxivity ratios and inference of MRI studies using agar phantom with different concentration of transition metal ions in EDA-functionalized TMCDs. Table S2 representing comparison of MR-imaging and optical imaging studies to determine apparent concentration of transition metal ions using agar phantom and in vivo zebrafish experiments. AUTHOR INFORMATION Corresponding Author *Tel: 91-413-2654975. Email: [email protected], [email protected] ‡†

Authors with equal contribution

ORCID Sajid Abdul Rub: 0000-0001-8011-497X. Shashank Shankar Chetty: 0000-0003-3563-2748. Praneetha Selvarasu: 0000-0001-8443-9333. Arumugam Vadivel Murugan: 0000-0003-4601-9615. Author Contributions P.S.A.R. performed the synthesis, analytical characterization and preparation of the manuscript with proper assist from S.S.C. Both S.S.C. and S.P. immensely helped in the interpretation of results and discussions. A.V.M. supervised and critically evaluated the entire research work. P.S.A.R carried out the in vitro cytotoxicity studies with the assistance of Y.K and P.L. as well as in vitro fluorescence imaging with the assistance of S.M and S.S.C. The MR imaging of agar


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phantom was carried out by P.S.A.R with the assistance of S.S.C. The agar phantom, in vivo optical and MR imaging with interpretation was carried out by S.S.C, P.S.A.R and K.S. All the authors have approved to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank the financial support provided by University Grants Commission (U.G.C)Maulana Azad National Fellowship, Ministry of Minority Affairs, Government of India. The facilities utilized from Central Instrumentation Facility (CIF), Pondicherry University to carry out material characterization are acknowledged. Dr. Udit B. Doss, Additional Medical Superintendent, Pondicherry Institute of Medical Sciences (PIMS) is accredited for his approval to conduct MRI and relaxometric studies. Finally, the assistance and inputs availed from the entire crew of MRI section in Pondicherry Institute of Medical Sciences - Mr. Oomadurai, Mr. Sudheendra, Mr. Pandiarajan, Mr. Leo Paul, Mr. Manikandan, Mr. Prince and Ms. Archana for interpreting the results of MRI analysis and relaxometric measurements were gratefully obliged. We also thank Dr. Mahadev and Ms. Anugraha for their help in carrying out MRI imaging in Apollo Super Speciality Cancer Hospital, Chennai.


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Transition Metal Ion (Mn2+, Fe2+, Co2+ and Ni2+)-Doped Carbon-dots Synthesized via Microwave-Assisted Pyrolysis: A Potential Nanoprobe for Magnetofluorescent Dual-Modality Bioimaging Sajid Abdul Rub Pakkath, ‡† Shashank Shankar Chetty, ‡† Praneetha Selvarasu, † Arumugam Vadivel Murugan, *† Yogesh Kumar, § Latha Periyasamy, § Muthukamalam Santhakumar, # Sudha Rani Sadras, # Kirankumar Santhakumar₸ Table of Contents: Rapid, microwave-assisted pyrolytic carbonization of lemon extract and subsequent doping of transition metal ions (Mn2+, Fe2+, Co2+ and Ni2+) to synthesize transition metal ion/C-dots as potential nanoprobes for magneto-fluorescent dual-modality bioimaging.


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Transition Metal Ion - ACS Publications

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